1. Field of the Invention
The present invention relates generally to unique metal binding proteins having high binding affinity for heavy metals. More particularly, the present invention is directed to compositions including the unique metal binding proteins and to associated methods of production and use where reduction or recovery of heavy metals is desired.
2. General Background and State of the Art
Metal recovery and metal remediation and the associated need for efficient and safe methods for clean up of metal waste is a continuing environmental and business concern due to the toxicity and potential risk to human health posed by metal contaminants, as well as the economic value of precious heavy metals. Indeed, as the discharge of toxic wastes such as heavy metals from agricultural, industrial and other commercial operations continues, the need for effective, safe and low-cost metal remediation methods increases. In a recent report by the U.S. EPA, metal contamination remains and historically has been a key concern at many contaminated sites (USEPA Work Assignment #011059, Mar. 5, 1997, Contract #68-W5-0055). In addition, there are numerous published reports of damage to wildlife, livestock, plantlife as well as danger to human health as a result of metal poisoning from contaminated soil or waste matter (Impact of Lead-Contaminated Soil on Public Health by Xintaras, C. May 1992 at http://www.atsdr.cdc.gov/cxlead.html). For example, a primary concern to humans is the health hazard created by lead (Pb) contamination. Exposure to lead can occur through a variety of methods such as by ingestion of lead from food, water, soil, or even inhalation of dust. Lead poisoning is extremely dangerous and potentially fatal, with symptoms including seizures, mental retardation and behavioral disorders. Therefore, methods for metal remediation are extremely valuable both for their protection of our environment as well as for protection from diseases.
Recovered metals from various waste, discard or recycling efforts provide immense economic value as well as augmenting environmental pollution control. Metal recovery can be from innumerable and varied sources such as from waste electronic devices (transistors, chips, transformers, bus bars, cathodes, and microprocessors, populated computer circuit boards PCBs, motherboards). Costs associated with hazardous disposal of industrial waste in the absence of metal reclamation are enormous. Therefore, metal recycling or reuse of metal extracted from scrap or discarded metal-containing items not only reduces the volume and cost of metal waste requiring specialized disposal and handling efforts, but the reclaimed metal can also be resold or reused to provide additional economic value.
Prior art attempts at treating metal contamination have traditionally employed cleanup technologies which consist primarily of physically removing and then disposing of contaminated matter. These methodologies are not only labor intensive and less efficient, but also carry a high expense associated with removal and disposal of large or bulk quantities of contaminated waste. Metal contamination is especially difficult to remediate because unlike other types of waste such as chemical or organic matter, metals cannot be directly destroyed or converted. For example, current technologies for remediating metal contaminated soils consist primarily of landfilling or soil excavation with physical or chemical separation of the metal contaminants. Treatment of contaminated ground water usually involves flushing, filtration or chemical extraction to remove the contaminating metals. As a result, the cost of soil or ground water remediation is high, ranging in the hundreds to thousands of millions of dollars in projected five-year costs per site (U.S. EPA, 1993).
In addition, the risk to humans and the environment from heavy metal contamination is not limited to soil or ground water, but also includes other sources such as industrial waste, sludge waste, wastewater, radionuclides (such as from research and medical waste) and mining waste. Depending on the physical and chemical form of the metal contaminant to be removed, as well as the cost-benefit analysis for a particular remediation approach, which of the existing technologies is better suited for a particular site will vary. However, due to the high cost of traditional cleanup technologies, there still remains a great need for a less-expensive, safe and effective heavy metal recovery and cleanup technology.
There are some technologies currently available for the recovery or remediation of heavy metal contaminated waste. In general, these technologies combine one or more of the following general approaches: isolation, immobilization, toxicity reduction, physical separation or extraction of metal contamination from a waste product. Isolation technologies utilize a containment strategy in an attempt to confine a contaminated site or area so as to prevent further spread of the toxic metal waste. Immobilization technologies reduce the mobility of metal contaminants and include systems which provide an impermeable barrier to separate underlying layers of soil (containing the metal contaminants) from the topsoil layer. Also used are physical barriers which restrict the flow of uncontaminated groundwater through a contaminated site. Additionally, there are toxicity reduction processes which generally use chemical or biological techniques to decrease the toxicity or mobility of metal contaminants. Included in toxicity reduction processes are biological treatment technologies, which apply newer biotechnical approaches.
Metal remediation is a relatively new application of biological treatment technologies and includes processes such as bioaccumulation, phytoremediation, phyotextraction, and rhizofiltration. All of these biological treatments use certain plants and microorganisms to remediate metals through either adsorption, absorption, or concentration of contaminating metal ions. For example, in bioaccumulation, plants or microorganisms actively take up and accumulate metals from contaminated surroundings.
In phytoremediation, specific plants that have developed the ability to selectively remove metal ions from soil are used. Such plants include certain xe2x80x9chyperaccumulatorxe2x80x9d species such as the alpine pennycrass plant, which is capable of accumulating metals at levels of 260 times greater than most plants before showing toxicity symptoms. Most hyperaccumulator plants, however, are very slow growing and have specific growth requirements. Some of these growth requirements are not conducive to the use of these plants at sites or in situations where metal recovery or remediation is needed. Furthermore, there are very few plant species known or available for recovery or remediation use. Therefore, given the persistent and high incidence of metal contamination at environmental and waste sites (xcx9c75% of Superfund Sites contain metal ions as a form of contamination, U.S. EPA, 1996), more efficient methods and approaches for removing heavy metals from contaminated sources are still needed.
More recently, in an attempt to meet these needs, biotechnological approaches have been employed as an alternative strategy to metal recovery and remediation. Included in these biotechnology approaches are the use of tobacco plants that have been manipulated to express metallothionein genes (Maiti et al., 1991). Metallothioneins (MTs) are small metal binding proteins ubiquitously distributed throughout the animal kingdom. They have high metal binding affinities and are believed to be important in controlling the intracellular levels of free metal ions. However, little else is known about their function or biological purpose. MTs were first discovered in 1957 in horse tissue. Since then, they have been identified in species ranging from fungi and shellfish to mice and humans.
The structural features of MTs include a high cysteine composition and lack of aromatic amino acids. The cysteine residues are responsible for the protein""s high affinity metal ion binding capabilities. In general, prior art MTs have a high degree of amino acid sequence similarity. However, the proteins or known gene sequences encoding the prior art proteins have been used primarily in either the research setting or in disease treatment methodologies.
Accordingly, one of the objects of the present invention is to provide novel metal binding proteins and associated methods for their production. This technology would allow for the efficient, cost effective, safe and simple removal of heavy metals from environmental waste or other materials contaminated with heavy metal.
Prior art metallothionein (MT) proteins are generally about 60-68 amino acid residues in size and have a high degree of sequence conservation among the different species. Whereas this high degree of sequence conservation and similarity contributed greatly to the ease of discovery of those MT genes, the novel metal binding proteins of the present invention differ substantially in sequence and were, therefore, much more difficult and required greater perseverance to obtain.
Unlike known prior art MTs, MTs from brine shrimp (Artemia) are much smaller in size (about 48 amino acid residues) and have distinctly unique amino acid and DNA sequences. As a result of these divergences in sequence from prior art MTs, prior to the present invention, the novel metal binding proteins of the present invention were extremely difficult to obtain and the nucleic acid sequences encoding these novel metal binding proteins unknown. The novel metal binding proteins of the present invention are capable of high capacity and high affinity metal binding. This makes them particularly suitable for use in pollution control, metal recycling, metal mining and other metal recovery and metal remediation technologies.
These and other objects are achieved by the compositions and methods of the present invention which provide for the efficient and reliable sequestration of heavy metals from a variety of sources. The novel metal binding proteins of the present invention can be expressed and produced easily for purposes such as metal remediation, metal recycling, metal mining or other types of processes where binding of one or more heavy metals is desired.
In accordance with the teachings of the present invention, novel metal binding proteins are provided. The invention includes at least one substantially purified metal binding protein having an amino acid sequence analogous to at least one metal binding protein sequence from brine shrimp (Artemia). A substantially purified metal binding protein can include an amino acid sequence such as:
and sequences incorporating one or more conservative amino acid substitutions of SEQ ID NO: 2 or SEQ ID NO:4. It should be noted that while the present invention will be discussed in the context of metal recovery and metal remediation, the present invention is readily applicable to many other uses where removal, recovery or simply binding of heavy metals is desired.
The metal binding proteins of the present invention also include a family of metal binding proteins having multiple isomeric forms. Accordingly, the family of metal binding proteins includes at least 5 isomeric forms of metal binding proteins. Any and all of these metal binding protein isomers are suitable for use in removal or recovery of heavy metals. The xe2x80x9cisomersxe2x80x9d of the present invention have the requisite structural features that classify them as metal binding proteins. These features include their high cysteine content, which confers their metal binding capacity. Therefore, the metal binding proteins of the present invention, including their isomeric forms, can be expressed and easily produced for purposes such as metal remediation, metal recycling, metal mining or other types of processes involving metal binding.
The novel metal binding proteins of the present invention also have characteristics that further enhance their use in the methods of the present invention and which further distinguish them from the prior art. These advantageous characteristics also render the novel metal binding proteins and associated methods particularly useful in a wide variety of metal recovery and metal remediation settings. For example, the metal binding proteins are capable of heavy metal binding under a range of conditions such as under moderate to high temperature conditions. Metal binding activity occurs from about 4xc2x0 C. to about 100xc2x0 C. Depending on a particular application or operation in which a metal binding protein of the present invention is to be implemented, a particular temperature range may be preferred. Therefore, in accordance with the present invention, the suitable range of temperatures include anywhere from about 4xc2x0 C. to about 100xc2x0 C. This range of temperature conditions which is incompatible with some prior art methodologies, renders our substantially purified metal binding proteins more versatile and preferable for use in metal recovery, metal remediation or other processes requiring heavy metal binding. Additionally, the metal binding proteins of the present invention are able to bind metal in a variety of chemical conditions. For example, metal binding activity occurs from about pH 4.0 to about 10.0. Bound metal ion(s) can be disassociated or removed from a metal binding protein of the present invention by lowering the pH to about 1.0. An exemplary method comprises slowly increasing the pH to about 7.0 in the presence of a reducing agent, such as dithiothreitol (DTT) for example. This reestablishes the metal binding activity of the metal binding protein and, therefore, the metal binding proteins can be reused if desired.
In further accordance with the teachings of the present invention, isolated nucleic acids encoding the metal binding proteins are provided. These isolated nucleic acids encode metal binding proteins having amino acid sequence analogous to at least one metal binding protein sequence from a brine shrimp (Artemia). An isolated nucleic acid of the present invention can include a DNA sequence such as:
Alternatively, an isolated nucleic acid of the present invention can include minimal DNA sequences which are sufficient to allow translation of a functional metal binding protein. A DNA sequence encoding a functional metal binding protein of the present invention need not comprise the entire native metal binding protein gene sequence but can be just those portions or regions of SEQ ID NO:1 that confer binding to heavy metals. For example, the present invention can include a DNA sequence comprising:
Additionally, the present invention also includes DNA having at least 80% or more sequence identity to a DNA molecule having the sequence of SEQ ID NO:1 or a DNA molecule having the sequence of nucleotide residues 1 to 66 of SEQ ID NO:1.
The isolated nucleic acids of the present invention also include nucleic acids encoding any and all of the isomeric or alternative forms of the metal binding proteins disclosed. Additionally, the isolated nucleic acids of the present invention need not comprise entire coding sequences of an MT isomer, but can include nucleic acid sequences encoding domains or portions of a coding sequence encoding an MT isomer, such as, for example, the functional or metal binding regions of the metal binding protein isomers of the present invention.
In further accordance with the teachings of the present invention, the novel metal binding proteins can be utilized as a naked composition or can be provided in association with a support, substrate, or other delivery system to aid in either the dispersal, handling, packaging or function of the metal binding proteins in metal recovery, metal remediation or metal binding processes as disclosed herein. Therefore, any of the metal binding proteins of the present invention can be coupled to a support such as a membrane or filter through which metal containing fluids are brought into contact.
The present invention is particularly well suited for use in metal recovery, metal remediation or metal recycling processes and methods. These methods include contacting a metal binding protein of the present invention having an amino acid sequence analogous to at least one metal binding protein sequence from brine shrimp (Artemia) with a substrate or material having a concentration of at least one heavy metal in order to bind the metal to the metal binding protein; and then separating the bound metal from the substrate or material.
For example, the methods of the present invention are useful in connection with the treatment of any substance having a concentration of at least one heavy metal. As will be appreciated by those skilled in the art, such heavy metal containing substances can be any environmental or industrial material such as ground water, drinking water, contaminated soil, waste, or the like, containing a concentration of metal. Similarly, the methods of the present invention are equally useful in treating industrial or municipal wastes containing metals that are desirable to remove. This broad utility makes the compositions and associated methods of the present invention particularly useful in a wide variety of circumstances.
In further accordance with the novel teachings of the present invention, expression systems producing the novel metal binding proteins of the present invention are provided. These novel metal binding proteins produced by the disclosed expression systems include the metal binding proteins having amino acid sequences analogous to at least one binding protein sequence from a brine shrimp (Artemia), as previously discussed. These expression systems include systems for the production or manufacturing of these compositions which can function in larger scale commercial or industrial plants, as well as in smaller scale, site-specific applications. Also included within the teachings of the present invention are expression systems of live or living production entities such as modified organisms and host cells. These include transgenic plants, transgenic animals and bacteria, and other modified organisms which have been genetically engineered to produce the novel metal binding proteins of the present invention.
Accordingly, the present invention provides unique, relatively small metal binding proteins having unique properties and sequences distinct from those of known prior art metal binding proteins. Furthermore, the novel metal binding proteins of the present invention retain high binding affinity for heavy metals in a variety of conditions, making them particularly useful in situations where removal or recovery of heavy metals from a substrate or any metal containing or metal contaminated source is desired. The novel metal binding proteins and the associated methods of the present invention provide for the efficient, cost effective, and safe removal and recovery of heavy metals from a wide variety of substrates.
The following Detailed Description provides additional enabling disclosure of the present invention and will make apparent to those skilled in the art additional features and advantages thereof.